Improved one-tube RT-PCR method for simultaneous detection and genotyping of duck hepatitis A virus subtypes 1 and 3

文献类型： 外文期刊

第一作者： Chen, Xueming

作者： Chen, Xueming;Chen, Yuhuan;Liu, Chungguo;Li, Xiaojun;Liu, Hongyu;Yin, Xiuchen;Bai, Xiaofei;Chen, Hongyan;Liu, Ming;Zhang, Yun;Chen, Yuhuan;Ge, Ming;Du, Yuanzhao;Fan, Gencheng

作者机构：

期刊名称：PLOS ONE （ 影响因子：3.24； 五年影响因子：3.788 ）

ISSN： 1932-6203

年卷期： 2019 年 14 卷 8 期

页码：

收录情况： SCI

摘要： Background The cocirculation of duck hepatitis A virus subtypes 1 (DHAV-1) and 3 (DHAV-3) in ducklings has resulted in significant economic losses. Ducklings with DHAV-1 or DHAV-3 infection show similar clinical signs and gross lesions; hence, it is important to identify the viral subtypes in infected ducklings as early as possible for better clinical management. Methods and results Based on multiple 5' noncoding region (5'-NCR) sequences of DHAV-1 and DHAV-3 strain alignments, universal and type-specific primers were designed and synthesized. With three primers in one-tube reverse transcription-PCR (RT-PCR), reference DHAV-1 and DHAV-3 isolates ranging over 60 years and across many different countries were successfully amplified, indicating that the primer sequences were completely conserved. The sequence results and the sizes of amplicons from reference DHAV-1 and DHAV-3 isolates are completely correlated with their subtypes. Moreover, with this one-tube RT-PCR system, amplicon sizes from liver samples of reference DHAV-1- or DHAV-3-infected birds fit closely with their subtypes, which was determined by virus isolation and neutralization testing. No other duck-origin RNA viruses were detected. The sensitivity of viral RNA detection was 10 pg. With this system, 20% subtype 1, 45% subtype 3, and 9% coinfection of two subtypes were detected in 55 clinical samples. Conclusions and significance This novel approach could be used for rapidly typing DHAV-1 or DHAV-3 infection in routine clinical surveillance or epidemiological screening.

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